PROJECT SUMMARY As the global population ages, developing interventions that can prevent or delay age-associated diseases is of increasing importance to promote healthy aging. As a result, there is intense interest in understanding the mechanisms by which calorie restriction (CR), an intervention which improves both lifespan and healthspan in wide range of model organisms, functions. Despite a century of study, the mechanism by which CR promotes health and longevity is still unknown. One prevailing model is that CR reduces the activity of mTOR Complex 1 (mTORC1), a protein kinase that is a key regulator of metabolism. Genetic and pharmaceutical inhibition of the mTORC1 signaling pathway extends lifespan, and studies in model organisms suggest an epistatic interaction, suggesting a key role for mTORC1 in the response to CR. However, studies from other labs have found that CR and mTORC1 inhibition have distinct effects on the transcriptome and metabolome other tissues, and the interaction of CR and reduced mTORC1 signaling on metabolism, health, and longevity have not been formally investigated in mammals. In fact, due to their very different feeding patterns, it is not even clear that CR-fed animals have a reduction in mTORC1 signaling relative to ad libitum (AL) controls. During our preliminary studies, we determined that mTORC1 signaling is similar in AL and CR fed fasted animals, and that CR-fed animals have elevated mTORC1 signaling as compared to AL-fed controls following feeding. We also determined that constitutively activating hepatic mTORC1 signaling does not block the metabolic effects of a CR diet. We also established that CR-fed animals are subject to both reduced calorie intake and prolonged daily fasting, as they consume their entire allotment of food within a period of ~2 hours, and fast for the remainder of the day, in sharp contrast to the normal food consumption pattern of a mouse. In preliminary experiments using different feeding regiments to separate out the effects of fasting and calorie intake, we have found that while many metabolic benefits of CR are mediated by calories, others may require fasting. The overall objective of the proposed project is to determine the role of mTORC1 in the mammalian response to CR and if the benefits of CR are mediated by a reduction in calories and/or prolonged daily fast. We will accomplish the objective with the following: 1) we will determine if CR-fed animals have reduced mTORC1 signaling over the course of a 24-hour cycle, and if reduced hepatic mTORC1 signaling is required in the metabolic response to CR using genetically modified mice; and 2) We will utilize a novel experimental paradigm to dissect fasting from calorie restriction in a calorie restriction paradigm, and longitudinally assess the effect of reduced caloric intake with and without the imposition of daily fasting on the metabolic health, frailty, and longevity of mice. Together, the proposed experiments will address long-standing biological questions regarding the mechanisms by which CR promotes metabolic health, fitness, and longevity, and will provided much needed insight into the role of mTORC1 in the mammalian response to CR.